STRUCTURE/FUNCTION OF EUKARYOTIC RNASE III

真核 RNA 酶 III 的结构/功能

• 批准号: 2024112
• 负责人: Manuel Ares
• 金额: $ 14万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2024112
• 关键词: cell growth regulation; enzyme mechanism; enzyme structure; enzyme substrate; gene expression; genetic enhancer element; molecular biology; protein structure function; ribonuclease III; ribosomal RNA; ribosomes; small nuclear RNA; suppressor mutations; yeasts

DESCRIPTION: The essential RNT1 (ribonuclease three) gene encodes a double strand-specific endoribonuclease from the yeast Saccharomyces cerevisiae. The RNT1 protein appears similar to bacterial RNase III in structure and function and is required for pre-ribosomal RNA processing events in yeast. Eukaryotic pre-rRNA processing is also dependent on small nucleolar RNAs, and at least one processing event is dependent on both yeast RNase III and U3 snoRNA. In bacteria, RNase III is involved in a wide variety of RNA processing events; thus the identification of the enzyme in yeast offers an unusual opportunity to explore the role of this fundamental enzyme in eukaryotic gene expression and RNA biogenesis, where it may influence mRNA stability or the replication of RNA viruses. An overarching hypothesis to be tested is that yeast RNase III is required for the synthesis or degradation of several unstable nonribosomal RNAs necessary for cell growth. In addition, the mechanism of snoRNA facilitation of RNase III activity deserves investigation. Three aims are proposed: 1) To characterize the structure, function and substrate specificity of the yeast RNase III enzyme using reverse genetics and biochemistry; 2) to identify and characterize substrates of RNase III whose processing is key to cell growth; and 3) To develop systems to study the mechanism by which snoRNAs function in concert with RNase III in pre-rRNA processing and ribosome assembly. The first specific aim will be addressed by studying the function of RNT1 protein and its mutants to determine which parts of the protein are required for substrate binding, cleavage, metal binding and multimerization. In the second specific aim genetic screens for suppressors and enhancers (synthetic lethal mutations) for two available temperature sensitive mutations in the RNT1 gene will be used to identify key substrates whose cleavage by RNase III is essential for cell growth. The third specific aim will require an effort to reproduce the observed in vivo requirement for both RNase III and the U3 snRNA in the A0 processing event in vitro. Together, the proposed experiments should provide an initial characterization of a conserved and fundamental eukaryotic gene product about which little is known.

描述：必需的RNT1(核糖核酸酶3)基因编码一个 酿酒酵母的链特异性内切核酸酶。 RNT1蛋白在结构上与细菌RNase III相似，并且 功能，是酵母中核糖体前RNA处理活动所必需的。 真核细胞的前rRNA加工也依赖于小核仁RNA， 并且至少一个处理事件依赖于酵母RNaseIII和 U3 snoRNA。在细菌中，RNaseIII参与了各种各样的RNA 处理事件；因此，酵母中酶的鉴定提供了一种 不同寻常的机会探索这种基础酶在 真核基因表达与RNA生物发生及其对mRNA的影响 RNA病毒的稳定性或复制。一个重要的假设是 需要检测的是酵母RNaseIII是合成或 细胞生长所必需的几种不稳定的非核糖体RNA的降解。 此外，snoRNA促进RNase III活性的机制 值得调查。 提出了三个目标：1)对其结构、功能和性能进行表征 用反向遗传学方法研究酵母核糖核酸酶III的底物特异性 和生化；2)核糖核酸酶III底物的鉴定和性质 其加工过程是细胞生长的关键；以及3)开发研究系统 SnoRNAs与RNaseIII协同工作的机制 前rRNA加工和核糖体组装。第一个具体目标将是 通过研究RNT1蛋白及其突变体的功能来解决 确定底物结合所需的蛋白质部分， 卵裂、金属结合和多聚体。在第二个具体目标中 对抑制子和增强子(合成致死突变)的基因筛选 对于两个可用的RNT1基因的温度敏感型突变 用于识别其被核糖核酸酶III切割的关键底物 细胞生长。第三个具体目标将需要努力再现 在体内观察到A0对RNaseIII和U3SnRNA的需求 在体外处理事件。总之，拟议的实验应该 提供了一个保守的和基本的 真核基因产物，人们对此知之甚少。